Inhibition of exoprotein production using aromatic compositions in non-absorbent articles

ABSTRACT

Non-absorbent articles are disclosed. The non-absorbent articles include an effective amount of an aromatic inhibitory compound to substantially inhibit the production of exotoxins by Gram positive bacteria. The aromatic inhibitory compounds of the present invention have the general formula:  
                 
 
     wherein R 1  is —OR 6 OH; R 6  is a divalent saturated or unsaturated aliphatic hydrocarbyl moiety; R 2 , R 3 , and R 4  are independently selected from the group consisting of H, OH, COOH, and —C(O)R 9 ; R 9  is hydrogen or a monovalent saturated or unsaturated aliphatic hydrocarbyl moiety.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This divisional patent application claims priority from U.S.patent application Ser. No. 09/969,391 filed on Oct. 2, 2001, theentirety of which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to the inhibition of exoproteinproduction in association with a non-absorbent article. Moreparticularly, the present invention relates to the coating or treatmentof certain aromatic compounds onto non-absorbent articles and theeffects of these compounds on Gram positive bacteria.

[0003] There exists in the female body a complex process which maintainsthe vagina and physiologically related areas in a healthy state. In afemale between the age of menarche and menopause, the normal vaginaprovides an ecosystem for a variety of microorganisms. Bacteria are thepredominant type of microorganism present in the vagina; most womenharbor about 10⁹ bacteria per gram of vaginal fluid. The bacterial floraof the vagina is comprised of both aerobic and anaerobic bacteria. Themore commonly isolated bacteria are Lactobacillus species,Corynebacteria, Gardnerella vaginalis, Staphylococcus species,Peptococcus species, aerobic and anaerobic Streptococcus species, andBacteroides species. Other microorganisms that have been isolated fromthe vagina on occasion include yeast (Candida albicans), protozoa(Trichomonas vaginalis), mycoplasma (Mycoplasma hominis), chlamydia(Chlamydia trachomatis), and viruses (Herpes simplex). These latterorganisms are generally associated with vaginitis or venereal disease,although they may be present in low numbers without causing symptoms.

[0004] Physiological, social, and idiosyncratic factors effect thequantity and species of bacteria present in the vagina. Physiologicalfactors include age, day of the menstrual cycle, and pregnancy. Forexample, vaginal flora present in the vagina throughout the menstrualcycle can include lactobacilli, corynebacterium, ureaplasma, andmycoplasma. Social and idiosyncratic factors include method of birthcontrol, sexual practices, systemic disease (e.g., diabetes), andmedications.

[0005] Bacterial proteins and metabolic products produced in the vaginacan effect other microorganisms and the human host. For example, thevagina between menstrual periods is mildly acidic having a pH rangingfrom about 3.8 to about 4.5. This pH range is generally considered themost favorable condition for the maintenance of normal flora. At thatpH, the vagina normally harbors the numerous species of microorganismsin a balanced ecology, playing a beneficial role in providing protectionand resistance to infection and makes the vagina inhospitable to somespecies of bacteria such as Staphylococcus aureus (S. aureus). The lowpH is a consequence of the growth of lactobacilli and their productionof acidic products. Microorganisms in the vagina can also produceantimicrobial compounds such as hydrogen peroxide and bactericidesdirected at other bacterial species. One example is the lactocins,bacteriocin-like products of lactobacilli directed against other speciesof lactobacilli.

[0006] Some microbial products produced in the vagina may negativelyaffect the human host. For example, S. aureus can produce and excreteinto its environment a variety of exoproteins including enterotoxins,Toxic Shock Syndrome Toxin-1 (TSST-1), and enzymes such as proteases andlipase. When absorbed into the bloodstream of the host, TSST-1 mayproduce Toxic Shock Syndrome (TSS) in non-immune humans.

[0007]S. aureus is found in the vagina of approximately 16% of healthywomen of menstrual age. Approximately 25% of the S. aureus isolated fromthe vagina are found to produce TSST-1. TSST-1 and some of thestaphylococcal enterotoxins have been identified as causing TSS inhumans.

[0008] Symptoms of Toxic Shock Syndrome generally include fever,diarrhea, vomiting and a rash followed by a rapid drop in bloodpressure. Multiple organ failure occurs in approximately 6% of those whocontract the disease. S. aureus does not initiate Toxic Shock Syndromeas a result of the invasion of the microorganism into the vaginalcavity. Instead as S. aureus grows and multiplies, it can produceTSST-1. Only after entering the bloodstream does TSST-1 toxin actsystemically and produce the symptoms attributed to Toxic ShockSyndrome.

[0009] Menstrual fluid has a pH of about 7.3. During menses, the pH ofthe vagina moves toward neutral and can become slightly alkaline. Thischange permits microorganisms whose growth is inhibited by an acidicenvironment the opportunity to proliferate. For example, S. aureus ismore frequently isolated from vaginal swabs during menstruation thanfrom swabs collected between menstrual periods.

[0010] When S. aureus is present in an area of the human body thatharbors a normal microbial population such as the vagina, it may bedifficult to eradicate the S. aureus bacterium without harming membersof the normal microbial flora required for a healthy vagina. Typically,antibiotics that kill S. aureus are not an option for use in productsinserted into the vagina because of their effect on the normal vaginalmicrobial flora and their propensity to stimulate toxin production ifall of the S. aureus are not killed. An alternative to completeeradication is technology designed to prevent or substantially reducethe bacterium's ability to produce toxins.

[0011] There have been numerous attempts to reduce or eliminatepathogenic microorganisms and menstrually occurring Toxic Shock Syndromeby incorporating one or more biostatic, biocidal, and/or detoxifyingcompounds into vaginal products. For example, L-ascorbic acid has beenapplied to a menstrual tampon to detoxify toxin found in the vagina.Others have incorporated monoesters and diesters of polyhydric aliphaticalcohols, such as glycerol monolaurate, as biocidal compounds (see,e.g., U.S. Pat. No. 5,679,369). Still others have introduced othernon-ionic surfactants, such as alkyl ethers, alkyl amines, and alkylamides as detoxifying compounds (see, e.g., U.S. Pat. Nos. 5,685,872,5,618,554, and 5,612,045).

[0012] Despite the aforementioned art, there continues to be a need forcompounds that will effectively inhibit the production of exoproteins,such as TSST-1, from Gram positive bacteria, and maintain activity evenin the presence of the enzymes lipase and esterase which can haveadverse effects on potency and which may also be present in the vagina.Further, it is desirable that the detoxifying compounds useful in theinhibition of the production of exoproteins be substantially non-harmfulto the natural flora found in the vaginal area. It is also desirablethat the detoxifying compound be coated or otherwise introduced onto anon-absorbent substrate prior to use.

SUMMARY OF THE INVENTION

[0013] The present invention is based on the discovery that when one ormore aromatic compounds having the general structure:

[0014] wherein R¹ is selected from the group consisting of H,

 —R⁶C(O)H, —R⁶OH, —R⁶COOH, —OR⁶OH, —OR⁶COOH, —C(O)NH₂,

[0015] and NH₂ and salts thereof; R⁵ is a monovalent saturated orunsaturated aliphatic hydrocarbyl moiety; R⁶ is a divalent saturated orunsaturated aliphatic hydrocarbyl moiety; R⁷ is a trivalent saturated orunsaturated aliphatic hydrocarbyl moiety; R⁸ is a monovalent substitutedor unsubstituted saturated or unsaturated aliphatic hydrocarbyl moietywhich may or may not be interrupted with hetero atoms; R², R³, and R⁴are independently selected from the group consisting of H, OH, COOH, and—C(O)R⁹; R⁹ is hydrogen or a monovalent saturated or unsaturatedaliphatic hydrocarbyl moiety, are incorporated onto a non-absorbentsubstrate, the production of exoprotein in Gram positive bacterium issubstantially inhibited.

[0016] The present invention relates to non-absorbent substrates orarticles which inhibit the production of exoproteins from Gram-positivebacteria. The substrates are particularly useful for inhibiting theproduction of TSST-1 from S. aureus bacteria in the vaginal area.Examples of suitable non-absorbent substrates which can have thearomatic compounds of the present invention incorporated thereon includenon-absorbent incontinence devices, barrier birth control devices,douches, contraceptive sponges, and tampon applicators. One specificexample of a non-absorbent incontinence device is a female barrierincontinence device, such as an incontinence pledget formed from aresilient material like rubber. Another suitable non-absorbent substrateis the applicator used with a tampon. For example, the tampon applicatormay have the aromatic compound coated on an outer surface, such thatwhen the applicator is used to introduce a tampon into a women's vaginathe aromatic compound (typically in the form of a cream, wax, gel orother suitable form) is transferred from the applicator onto the wall ofthe vagina.

[0017] It is a general object of the present invention to provide anon-absorbent article which inhibits the production of exoprotein fromGram positive bacterium. A more specific object of the present inventionis to provide a non-absorbent incontinence device, a barrier birthcontrol device, a contraceptive sponge, tampon applicator, or a doucheincorporating one or more aromatic compounds which act to substantiallyinhibit the production of TSST-1 and Enterotoxin B by S. aureus.

[0018] Another object of the present invention is to provide anon-absorbent substrate incorporating one or more aromatic compounds incombination with one or more other inhibitory ingredients such as, butnot limited to, for example, laureth-4, PPG-5 lauryl ether,1-0-dodecyl-rac-glycerol, disodium laureth sulfosuccinate, glycerolmonolaurate, alkylpolyglycosides, polyethylene oxide (2) sorbital etheror myreth-3-myristate which in combination act to substantially inhibitthe production of TSST-1 and Enterotoxin B by S. aureus.

[0019] A further object of the present invention is to provide anon-absorbent substrate that has incorporated therewith one or morecompounds that will inhibit the production of exoproteins from Grampositive bacterium without significantly imbalancing the natural florapresent in the vaginal tract.

[0020] Other objects and advantages of the present invention, andmodifications thereof, will become apparent to persons skilled in theart without departure from the inventive concepts defined in the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] In accordance with the present invention, it has been discoveredthat aromatic compounds as described herein can be used in combinationwith non-absorbent articles, such as incontinence devices, for example,to substantially inhibit the production of exoproteins, such as TSST-1,from Gram positive bacteria. It has also been discovered that thearomatic compounds can also be used in combination with surface-activeagents such as, for example, compounds with an ether, ester, amide,glycosidic, or amine bond linking a C₈-C₁₈ fatty acid to an aliphaticalcohol, polyalkoxylated sulfate salt, or polyalkoxylated sulfosuccinicsalt, to substantially inhibit the production of exoproteins such asTSST-1 from Gram positive bacteria.

[0022] This invention will be described herein in detail in connectionwith various non-absorbent substrates or products such as non-absorbentincontinence devices, barrier birth control devices, contraceptivesponges, tampon applicators, and douches, but will be understood bypersons skilled in the art to be applicable to other non-absorbentarticles, devices and/or products as well wherein the inhibition ofexoproteins from Gram positive bacteria would be beneficial. As usedherein, the phrase “non-absorbent article” generally refers tosubstrates or devices which include an outer layer formed from asubstantially hydrophobic material which repels fluids such as menses,blood products and the like. Suitable materials for construction thenon-absorbent articles of the present invention include, for example,rubber, plastic, and cardboard.

[0023] It has been discovered that certain aromatic compounds cansubstantially inhibit the production of exoprotein by Gram positivebacterium and, specifically, the production of TSST-1 and Enterotoxin Bfrom S. aureus bacterium. The aromatic compounds useful in the presentinvention have the general chemical structure:

[0024] wherein R¹ is selected from the group consisting of H,

 —R⁶C(O)H, —R⁶OH, —R⁶COOH, —OR⁶OH, —OR⁶COOH, —C(O)NH₂,

[0025] and NH2 and salts thereof; R⁵ is a monovalent saturated orunsaturated aliphatic hydrocarbyl moiety; R⁶ is a divalent saturated orunsaturated aliphatic hydrocarbyl moiety; R⁷ is a trivalent saturated orunsaturated aliphatic hydrocarbyl moiety; R⁸ is hydrogen or a monovalentsubstituted or unsubstituted saturated or unsaturated aliphatichydrocarbyl moiety which may or may not be interrupted with heteroatoms; R², R³, and R⁴ are independently selected from the groupconsisting of H, OH, COOH, and —C(O)R⁹; R⁹ is a monovalent saturated orunsaturated aliphatic hydrocarbyl moiety.

[0026] The hydrocarbyl moieties described herein include both straightchain and branched chain hydrocarbyl moieties and may or may not besubstituted and/or interrupted with hetero atoms. Desirably, thearomatic compounds for use in the present invention contain at least oneOH and/or COOH group. The OH and/or COOH group can be bonded to thearomatic structure, or can be bonded to an atom which may or may not bedirectly bonded to the aromatic structure. R⁵ is desirably a monovalentsaturated aliphatic hydrocarbyl moiety having from 1 to about 15 carbonatoms, desirably from 1 to about 14 carbon atoms. R⁶ is desirably adivalent saturated or unsaturated aliphatic hydrocarbyl moiety havingfrom 1 to about 15 carbon atoms, desirably from 1 to about 14 carbonatoms. R⁷ is desirably a trivalent saturated or unsaturated aliphatichydrocarbyl moiety having from 1 to about 15 carbon atoms, desirablyfrom 1 to about 10 carbon atoms, and more desirably from 1 to about 4carbon atoms. Hetero atoms which can interrupt the hydrocarbyl moietyinclude, for example, oxygen and sulfur.

[0027] Preferred aromatic compounds of the present invention include2-phenylethanol, benzyl alcohol, trans-cinnamic acid, 4-hydroxybenzoicacid, methyl ester, 2-hydroxybenzoic acid, 2-hydoxybenzamide, acetyltyrosine, 3, 4,5-trihydroxybenzoic acid, lauryl3,4,5-trihydroxybenzoate, phenoxyethanol, 4-hydroxy-3-methoxybenzoicacid, p-aminobenzoic acid, and 4-acetamidophenol.

[0028] In accordance with the present invention, the non-absorbentarticle including the aromatic compound contains an effective amount ofthe inhibiting aromatic compound to substantially inhibit the formationof TSST-1 when the non-absorbent article or inhibiting compound thereonis exposed to S. aureus bacteria. Several methods are known in the artfor testing the effectiveness of potential inhibitory agents on theinhibition of the production of TSST-1 in the presence of S. aureus. Onesuch preferred method is set forth in Example 1 below. When tested inaccordance with the testing methodology described herein, desirably, theinhibiting aromatic compounds reduce the formation of TSST-1 when thenon-absorbent article is exposed to S. aureus by at least about 40%,more desirably by at least about 50%, still more desirably by at leastabout 60%, still more desirably by at least about 70%, still moredesirably by at least about 80%, still more desirably by at least about90%, and still more desirably by at least about 95%.

[0029] Effective amounts of aromatic compound that significantly reducethe production of TSST-1 have been found to be at least about 0.1micromoles of the aromatic compound per gram of the non-absorbentproduct. Desirably, the aromatic compound ranges from about 0.5micromoles per gram of non-absorbent to about 100 micromoles per gram ofnon-absorbent and more desirably from about 1.0 micromoles per gram ofnon-absorbent to about 50 micromoles per gram of non-absorbent. Although“aromatic compound” is used in the singular, one skilled in the artwould understand that it includes the plural, and that various aromaticcompounds within the scope of this invention may be used in combination.

[0030] The aromatic compounds of the present invention can be preparedand applied to the non-absorbent article in any suitable form, but aretypically prepared in forms including, without limitation, aqueoussolutions, lotions, balms, gels, salves, ointments, boluses,suppositories, and the like. One skilled in the art would recognize thatother forms may perform equally well.

[0031] The aromatic compounds may be applied to the non-absorbentarticle using conventional methods for applying an inhibitory agent tothe desired non-absorbent article. For example, non-absorbent articlesmay be dipped directly into a liquid bath having the inhibitory compoundand then can be air dried, if necessary, to remove any volatilesolvents. Alternatively, the non-absorbent articles of the presentinvention can be sprayed or otherwise coated with the inhibitoryaromatic compounds of the present invention.

[0032] The substantially inhibitory aromatic compounds may additionallyemploy one or more conventional pharmaceutically-acceptable andcompatible carrier materials useful for the desired application. Thecarrier can be capable of co-dissolving or suspending the materials usedon the non-absorbent article. Carrier materials suitable for use in theinstant invention include those well-known for use in the cosmetic andmedical arts as a basis for ointments, lotions, creams, salves,aerosols, suppositories, gels, and the like. For example, the aromaticcompound can be formulated into a variety of formulation such as thoseemployed in current commercial douche formulations, or in higherviscosity douches.

[0033] The aromatic compounds of the present invention may additionallyemploy adjunct components conventionally found in pharmaceuticalcompositions in their art-established fashion and at theirart-established levels. For example, the compositions may containadditional compatible pharmaceutically active materials for combinationtherapy, such as supplementary antimicrobial, antioxidants,anti-parasitic agents, antipruritics, astringents, local anaesthetics,or anti-inflammatory agents.

[0034] In another embodiment of the present invention, the inhibitoryaromatic compounds described above can be used in combination with oneor more surface active agents to reduce the production of TSST-1 withoutsignificantly eliminating the beneficial bacterial flora. The surfaceactive agents can include, for example, compounds with an ether, ester,amide, glycosidic, or amine bond linking a C₈-C₁₈ fatty acid to analiphatic alcohol, polyalkoxylated sulfate salt, or polyalkoxylatedsulfosuccinic salt.

[0035] In one embodiment, the inhibitory aromatic compounds describedherein can be used in combination with ether compounds having thegeneral formula:

[0036] wherein R¹⁰ is a straight or branched alkyl or alkenyl grouphaving a chain of from about 8 to about 18 carbon atoms and R¹¹ isselected from an alcohol, a polyalkoxylated sulfate salt or apolyalkoxylated sulfosuccinate salt.

[0037] The alkyl, or the R¹⁰ moiety of the ether compounds useful foruse in combination with the inhibitory aromatic compounds describedherein, can be obtained from saturated and unsaturated fatty acidcompounds. Suitable compounds include, C₈-C₁₈ fatty acids, andpreferably, fatty acids include, without limitation, caprylic, capric,lauric, myristic, palmitic and stearic acid whose carbon chain lengthsare 8, 10, 12, 14, 16, and 18, respectively. Highly preferred materialsinclude capric, lauric, and myristic acids.

[0038] Preferred unsaturated fatty acids are those having one or twocis-type double bonds and mixtures of these materials. Suitablematerials include myrystoleic, palmitoleic, linolenic and mixturesthereof.

[0039] Desirably, the R¹¹ moiety is an aliphatic alcohol which can beethoxylated or propoxylated for use in the ether compositions incombination with the inhibitory aromatic compounds described herein.Suitable aliphatic alcohols include glycerol, sucrose, glucose, sorbitoland sorbitan. Preferred ethoxylated and propoxylated alcohols includeglycols such as ethylene glycol, propylene glycol, polyethylene glycoland polypropylene glycol.

[0040] The aliphatic alcohols can be ethoxylated or propoxylated byconventional ethoxylating or propoxylating compounds and techniques. Thecompounds are preferably selected from the group consisting of ethyleneoxide, propylene oxide, and mixtures thereof, and similar ringedcompounds which provide a material which is effective.

[0041] The R¹¹ moiety can further include polyalkoxylated sulfate andpolyalkoxylated sulfosuccinate salts. The salts can have one or morecations. Preferably, the cations are sodium, potassium or both.

[0042] Preferred ether compounds for use in combination with theinhibitory aromatic compounds described herein include laureth-3,laureth-4, laureth-5, PPG-5 lauryl ether, 1-0-dodecyl-rac-glycerol,sodium laureth sulfate, potassium laureth sulfate, disodium laureth (3)sulfosuccinate, dipotassium laureth (3) sulfosuccinate, and polyethyleneoxide (2) sorbitol ether.

[0043] In accordance with the present invention, the non-absorbentarticle contains an effective amount of the combination of theinhibitory aromatic and ether compounds. The amount of ether compoundintroduced onto the non-absorbent article is at least about 0.0001millimoles of ether compound per gram of non-absorbent article, anddesirably at least about 0.005 millimoles of ether compound per gram ofnon-absorbent article. In a preferred embodiment, the non-absorbentarticle contains from about 0.005 millimoles of ether compound per gramof non-absorbent article to about 2 millimoles of ether compound pergram of non-absorbent article.

[0044] The non-absorbent articles of the present invention containing acombination of two active ingredients can be a variety of non-absorbentarticles including, for example, incontinence devices, barrier birthcontrol devices, contraceptive sponges, douches, tampon applicators, andthe like.

[0045] The non-absorbent articles of the present invention containing afirst inhibitory aromatic compound and a second inhibitory ethercompound contain a sufficient amount of both inhibitory compounds tosubstantially inhibit the formation of TSST-1 when the non-absorbentarticle is exposed to S. aureus bacteria. Desirably, the combination ofinhibitory compounds reduces the formation of TSST-1 when thenon-absorbent article is exposed to S. aureus by at least about 40%,more desirably at least about 50%, still more desirably at least about60%, still more desirably by at least about 70%, still more desirably byat least about 80%, still more desirably by at least about 90%, andstill more desirably by at least about 95%.

[0046] The non-absorbent articles of the present invention containingthe combination of aromatic inhibitory compounds and ether inhibitorycompounds may additionally employ adjunct components conventionallyfound in pharmaceutical compositions in their art-established fashionand at their art-established levels. For example, the compositions maycontain additional compatible pharmaceutically active materials forcombination therapy, such as supplementary antimicrobial, antioxidants,anti-parasitic agents, antipruritics, astringents, local anaesthetics,or anti-inflammatory agents.

[0047] Typically, the non-absorbent article will contain a molar ratioof inhibitory aromatic compound to ether compound of from about 1:6 toabout 1:0.05.

[0048] In another embodiment, the inhibitory aromatic compoundsdescribed herein can be used in combination with an alkyl polyglycosidecompound. Suitable alkyl polyglycosides for use in combination with theinhibitory aromatic compounds include alkyl polyglycosides having thegeneral formula:

[0049] wherein Z is a saccharide residue having 5 or 6 carbon atoms, nis a whole number from 1 to 6, and R¹⁴ is a linear or branched alkylgroup having from about 8 to about 18 carbon atoms. Commerciallyavailable examples of suitable alkyl polyglycosides having differingcarbon chain lengths include Glucopon 220, 225, 425, 600, and 625, allavailable from Henkel Corporation (Ambler, Pa.). These products are allmixtures of alkyl mono- and oligoglucopyranosides with differing alkylgroup chain lengths based on fatty alcohols derived from coconut and/orpalm kernel oil. Glucopon 220, 225, and 425 are examples of particularlysuitable alkyl polyglycosides for use in combination with the inhibitoryaromatic compounds of the present invention. Another example of asuitable commercially available alkyl polyglycoside is TL 2141, aGlucopon 220 analog available from ICI Surfactants (Wilmington, Del.).

[0050] It should be understood that as referred to herein, analkylpolyglycoside may consist of a single type of alkyl polyglycosidemolecule or, as is typically the case, may include a mixture ofdifferent alkyl polyglycoside molecules. The different alkylpolyglycoside molecules may be isomeric and/or may be alkylpolyglycoside molecules with differing alkyl group and/or saccharideportions. By use of the term alkyl poyglycoside isomers reference ismade to alkyl polyglycosides which, although including the same alkyether residues, may vary with respect to the location of the alkyl etherresidue in the alkyl polyglycoside as well as isomers which differ withrespect to the orientation of the functional groups about one or morechiral centers in the molecules. For example, an alkyl polyglycoside caninclude a mixture of molecules with saccharide portions which are mono,di-, or oligosaccharides derived from more than one 6 carbon saccharideresidue and where the mono-, di- or oligosaccharide has been etherifiedby reaction with a mixture of fatty alcohols of varying carbon chainlength. The present alkyl polyglycosides desirably include alkyl groupswhere the average number of carbon atoms in the alkyl chain is about 8to about 12. One example of a suitable alkyl polyglycoside is a mixtureof alkyl polyglycoside molecules with alkyl chains having from about 8to about 10 carbon atoms.

[0051] The alkyl polyglycosides employed in the non-absorbent articlesin combination with the inhibiting aromatic compounds can becharacterized in terms of their hydrophilic lipophilic balance (HLB).This can be calculated based on their chemical structure usingtechniques well known to those skilled in the art. The HLB of the alkylpolyglycosides used in the present invention typically falls within therange of about 10 to about 15. Desirably, the present alkylpolyglycosides have an HLB of at least about 12 and, more desirably,about 12 to about 14.

[0052] In accordance with the present invention, the non-absorbentarticle contains an effective amount of the combination of theinhibitory aromatic and alkyl polyglycoside compounds. The amount ofalkyl polyglycoside compound included in the non-absorbent article is atleast about 0.0001 millimoles of alkyl polyglycoside per gram ofnon-absorbent article, and desirably at least about 0.005 millimoles ofalkyl polyglycoside per gram of non-absorbent article. In a preferredembodiment, the non-absorbent article contains from about 0.005millimoles per gram of non-absorbent article to about 2 millimoles pergram of non-absorbent article.

[0053] The non-absorbent articles of the present invention containing acombination of inhibitory or active ingredients such as aromaticinhibitory compounds and alkyl polyglycoside inhibitory compounds can bea variety of non-absorbent articles including, for example, incontinencedevices, barrier birth control devices, contraceptive sponges, douches,tampon applicators, and the like.

[0054] The non-absorbent articles of the present invention containing afirst inhibitory aromatic compound and a second inhibitory alkylpolyglycoside compound contain a sufficient amount of both inhibitorycompounds to substantially inhibit the formation of TSST-1 when thenon-absorbent article is exposed to S. aureus bacteria. Desirably, thecombination of inhibitory compounds reduces the formation of TSST-1 whenthe non-absorbent article is exposed to S. aureus by at least about 40%,more desirably at least about 50%, still more desirably at least about60%, still more desirably by at least about 70%, still more desirably byat least about 80%, still more desirably by at least about 90%, andstill more desirably by at least about 95%.

[0055] The non-absorbent articles of the present invention containingthe combination of aromatic inhibitory compounds and alkyl polyglycosideinhibitory compounds may additionally employ adjunct componentsconventionally found in pharmaceutical compositions in theirart-established fashion and at their art-established levels. Forexample, the compositions may contain additional compatiblepharmaceutically active materials for combination therapy, such assupplementary antimicrobial, antioxidants, anti-parasitic agents,antipruritics, astringents, local anaesthetics, or anti-inflammatoryagents.

[0056] Typically, the non-absorbent article will contain a molar ratioof inhibitory aromatic compound to alkyl glycoside compound of fromabout 1:1 to about 1:0.05.

[0057] In another embodiment, the inhibitory aromatic compoundsdescribed herein can be used in combination with an amide containingcompound having the general formula:

[0058] wherein R¹⁷, inclusive of the carbonyl carbon, is an alkyl grouphaving 8 to 18 carbon atoms, and R¹⁸ and R¹⁹ are independently selectedfrom hydrogen or an alkyl group having from 1 to about 12 carbon atomswhich may or may not be substituted with groups selected from estergroups, ether groups, amine groups, hydroxyl groups, carboxyl groups,carboxyl salts, sulfonate groups, sulfonate salts, and mixtures thereof.

[0059] R¹⁷ can be derived from saturated and unsaturated fatty acidcompounds. Suitable compounds include, C₈-C₁₈ fatty acids, andpreferably, the fatty acids include, without limitation, caprylic,capric, lauric, myristic, palmitic and stearic acid whose carbon chainlengths are 8, 10, 12, 14, 16, and 18, respectively. Highly preferredmaterials include capric, lauric, and myristic.

[0060] Preferred unsaturated fatty acids are those having one or twocis-type double bonds and mixtures of these materials. Suitablematerials include myrystoleic, palmitoleic, linolenic and mixturesthereof.

[0061] The R¹⁸ and R¹⁹ moieties can be the same or different and eachbeing selected from hydrogen and an alkyl group having a carbon chainhaving from 1 to about 12 carbon atoms. The R¹⁸ and R¹⁹ alkyl groups canbe straight or branched and can be saturated or unsaturated. When R¹⁸and/or R¹⁹ are an alkyl moiety having a carbon chain of at least 2carbons, the alkyl group can include one or more substituent groupsselected from ester, ether, amine, hydroxyl, carboxyl, carboxyl salts,sulfonate and sulfonate salts. The salts can have one or more cationsselected from sodium, potassium or both.

[0062] Preferred amide compounds for use in combination with theinhibitory aromatic compounds described herein include sodium laurylsarcosinate, lauramide monoethanolamide, lauramide diethanolamide,lauramidopropyl dimethylamine, disodium lauramido monoethanolamidesulfosuccinate and disodium lauroamphodiacetate.

[0063] In accordance with the present invention, the non-absorbentarticle contains an effective amount of the combination of theinhibitory aromatic and amide-containing compounds. The amount ofamide-containing compound included in the non-absorbent article is atleast about 0.0001 millimoles of nitrogen containing compound per gramof non-absorbent article, and desirably at least about 0.005 millimolesof nitrogen containing compound per gram of non-absorbent article. In apreferred embodiment, the non-absorbent article contains from about0.005 millimoles per gram of non-absorbent article to about 2 millimolesper gram of non-absorbent article.

[0064] The non-absorbent articles of the present invention containing acombination of inhibitory or active ingredients such as aromaticinhibitory compounds and amide-containing inhibitory compounds can be avariety of non-absorbent articles including, for example, incontinencedevices, barrier birth control devices, contraceptive sponges, douches,tampon applicators, and the like.

[0065] The non-absorbent articles of the present invention containing afirst inhibitory aromatic compound and a second inhibitoryamide-containing compound contain a sufficient amount of both inhibitorycompounds to substantially inhibit the formation of TSST-1 when thenon-absorbent article is exposed to S. aureus bacteria. Desirably, thecombination of inhibitory compounds reduces the formation of TSST-1 whenthe non-absorbent article is exposed to S. aureus by at least about 40%,more desirably at least about 50%, still more desirably at least about60%, still more desirably by at least about 70%, still more desirably byat least about 80%, still more desirably by at least about 90%, andstill more desirably by at least about 95%.

[0066] The non-absorbent articles of the present invention containingthe combination of aromatic inhibitory compounds and amide-containinginhibitory compounds may additionally employ adjunct componentsconventionally found in pharmaceutical compositions in theirart-established fashion and at their art-established levels. Forexample, the compositions may contain additional compatiblepharmaceutically active materials for combination therapy, such assupplementary antimicrobial, antioxidants, anti-parasitic agents,antipruritics, astringents, local anaesthetics, or anti-inflammatoryagents.

[0067] Typically, the non-absorbent article will contain a molar ratioof inhibitory aromatic compound to amide-containing compound of fromabout 1:2 to about 1:0.05.

[0068] In another embodiment, the inhibitory compounds described hereincan be used in combination with amine compounds having the generalformula:

[0069] wherein R²⁰ is an alkyl group having from about 8 to about 18carbon atoms and R²¹ and R²² are independently selected from the groupconsisting of hydrogen and alkyl groups having from 1 to about 18 carbonatoms and which can have one or more substitutional moieties selectedfrom the group consisting of hydroxyl, carboxyl, carboxyl salts andimidazoline The combination of aromatic compounds and amine compoundsare effective in substantially inhibiting the production of exoproteinfrom Gram positive bacteria.

[0070] Desirably, R²⁰ is derived from fatty acid compounds whichinclude, without limitation, caprylic, capric, lauric, myristic,palmitic and stearic acid whose carbon chain lengths are 8, 10, 12, 14,16, and 18, respectively. Highly preferred materials include capric,lauric, and myristic. Preferred unsaturated fatty acids are those havingone or two cis-type double bonds and mixtures of these materials.Suitable materials include myrystoleic, palmitoleic, linolenic, andmixtures thereof.

[0071] The R²¹ and R²² alkyl groups can further include one or moresubstitutional moieties selected from hydroxyl, carboxyl, carboxylsalts, and R¹ and R² can form an unsaturated heterocyclic ring thatcontains a nitrogen that connects via a double bond to the alpha carbonof the R¹ moiety to form a substituted imidazoline. The carboxyl saltscan have one or more cations selected from sodium potassium or both. TheR²⁰, R²¹, and R²² alkyl groups can be straight or branched and can besaturated or unsaturated.

[0072] Preferred amine compounds for use with the aromatic compoundsdescribed herein include triethanolamide laureth sulfate, lauramine,lauramino propionic acid, sodium lauriminodipropionic acid, laurylhydroxyethyl imidazonline and mixtures thereof.

[0073] In another embodiment, the amine compound can be an amine salthaving the general formula:

[0074] wherein R²³ is an anionic moiety associated with the amine and isderived from an alkyl group having from about 8 to about 18 carbonatoms, and R²⁴, R²⁵, and R²⁶ are independently selected from the groupconsisting of hydrogen and alkyl group having from 1 to about 18 carbonatoms and which can have one or more substitutional moieties selectedfrom the group consisting of hydroxyl, carboxyl, carboxyl salts, andimidazoline. R²⁴, R²⁵, and R²⁶ can be saturated or unsaturated.Desirably, R²³ is a polyalkyloxylated alkyl sulfate. A preferredcompound illustrative of an amine salt is triethanolamide laurethsulfate.

[0075] In accordance with the present invention, the non-absorbentarticle contains an effective amount of the combination of theinhibitory aromatic and amine and/or amine salt compounds. The amount ofamine and/or amine salt compound included in the non-absorbent articleis at least about 0.0001 millimoles of ether per gram of non-absorbentarticle, and desirably at least about 0.005 millimoles of ether per gramof non-absorbent article. In a preferred embodiment, the non-absorbentarticle contains from about 0.005 millimoles per gram of non-absorbentarticle to about 2 millimoles per gram of non-absorbent article.

[0076] The non-absorbent articles of the present invention containing acombination of two active ingredients can be a variety of non-absorbentarticles including, for example, incontinence devices, barrier birthcontrol devices, contraceptive sponges, douches, tampon applicators, andthe like.

[0077] The non-absorbent articles of the present invention containing afirst inhibitory aromatic compound and a second inhibitory amine and/oramine salt compound contain a sufficient amount of both inhibitorycompounds to substantially inhibit the formation of TSST-1 when thenon-absorbent article is exposed to S. aureus bacteria. Desirably, thecombination of inhibitory compounds reduces the formation of TSST-1 whenthe non-absorbent article is exposed to S. aureus by at least about 40%,more desirably at least about 50%, still more desirably at least about60%, still more desirably by at least about 70%, still more desirably byat least about 80%, still more desirably by at least about 90%, andstill more desirably by at least about 95%.

[0078] The non-absorbent articles of the present invention containingthe combination of aromatic inhibitory compounds and amine and/or aminesalt inhibitory compounds may additionally employ adjunct componentsconventionally found in pharmaceutical compositions in theirart-established fashion and at their art-established levels. Forexample, the compositions may contain additional compatiblepharmaceutically active materials for combination therapy, such assupplementary antimicrobial, antioxidants, anti-parasitic agents,antipruritics, astringents, local anaesthetics, or anti-inflammatoryagents.

[0079] Typically, the non-absorbent article will contain a molar ratioof inhibitory aromatic compound to amine and/or amine salt compound offrom about 1:2 to about 1:0.05.

[0080] The present invention is illustrated by the following exampleswhich are merely for the purpose of illustration and are not to beregarded as limiting the scope of the invention or manner in which itmay be practiced.

EXAMPLE 1

[0081] In this Example, the effect of various test compounds on thegrowth of S. aureus and the production of TSST-1 was determined. Thetest compound, in the desired concentration (expressed in percent ofactive compound) was placed in 10 mL of a growth medium in a sterile, 50mL conical polypropylene tube (Sarstedt, Inc. Newton, N.C.).

[0082] The growth medium was prepared by dissolving 37 grams of brainheart infusion broth (BHI) (Difco Laboratories, Cockeysville, Md.) in880 mL of distilled water and sterilizing the broth according to themanufacturer's instructions. The BHI was supplemented with fetal bovineserum (FBS) (100 mL) (Sigma Chemical Company, St. Louis, Mo.).Hexahydrate of magnesium chloride (0.021 M, 10 mL) (Sigma ChemicalCompany, St. Louis, Mo.) was added to the BHI-FBS mixture. Finally,L-glutamine (0.027 M, 10 mL) (Sigma Chemical Company, St. Louis, Mo.)was added to the mixture.

[0083] Compounds to be tested included phenylethyl alcohol, benzylalcohol, and 2-hydroxybenzamide. Test compounds were both liquids andsolids. The liquid test compounds were added directly to the growthmedium and diluted in growth medium to obtain the desired finalconcentrations. The solid test concentrations were dissolved inmethanol, spectrophotometric grade (Sigma Chemical Company, St. Louis,Mo.) at a concentration that permitted the addition of 200 microlitersof the solution to 10 mL of growth medium for the highest concentrationtested. Each test compound that was dissolved in methanol was added tothe growth medium in the amount necessary to obtain the desired finalconcentration.

[0084] In preparation for inoculation of the tubes of growth mediumcontaining the test compounds, an inoculating broth was prepared asfollows: S. aureus (MN8) was streaked onto a tryptic soy agar plate(TSA; Difco Laboratories Cockeysville, Md.) and incubated at 35° C. Thetest organism was obtained from Dr. Pat Schlievert, Department ofMicrobiology, University of Minnesota Medical School, Minneapolis, Minn.After 24 hours of incubation three to five individual colonies werepicked with a sterile inoculating loop and used to inoculate 10 mL ofgrowth medium. The tube of inoculated growth medium was incubated at 35°C. in atmospheric air. After 24 hours of incubation, the culture wasremoved from the incubator and mixed well on a S/P brand vortex mixer. Asecond tube containing 10 mL of the growth medium was inoculated with0.5 mL of the above-described 24 hour old culture and incubated at 35°C. in atmospheric air. After 24 hours of incubation the culture wasremoved from the incubator and mixed well on a S/P brand vortex mixer.The optical density of the culture fluid was determined in a microplatereader (Bio-Tek Instruments, Model EL309, Winooski, Vt.). The amount ofinoculum necessary to give 5×10⁶ CFU/mL in 10 mL of growth medium wasdetermined using a standard curve.

[0085] This Example included tubes of growth medium with varyingconcentrations of test compounds, tubes of growth medium without testcompounds (control) and tubes of growth medium with 20-400 microlitersof methanol (control). Each tube was inoculated with the amount ofinoculum determined as described above. The tubes were capped with foamplugs (Identi-plug plastic foam plugs, Jaece Industries purchased fromVWR Scientific Products, South Plainfield, N.J.). The tubes wereincubated at 35° C. in atmospheric air containing 5% by volume CO₂.After 24 hours of incubation the tubes were removed from the incubatorand the optical density (600 nm) of the culture fluid was determined andthe culture fluid was assayed for the number of colony forming units ofS. aureus and was prepared for the analysis of TSST-1 as describedbelow.

[0086] The number of colony forming units per mL after incubation wasdetermined by standard plate count procedures. In preparation foranalysis of TSST-1, the culture fluid broth was centrifuged and thesupernatant subsequently filter sterilized through an Autovial 5syringeless filter, 0.2 micrometers pore size (Whatman, Inc., Clifton,N.J.). The resulting fluid was frozen at −70° C. until assayed.

[0087] The amount of TSST-1 per mL was determined by a non-competitive,sandwich enzyme-linked immunonon-absorbent assay (ELISA). Samples of theculture fluid and the TSST-1 reference standard were assayed intriplicate. The method employed was as follows: four reagents, TSST-1(#TT-606), rabbit polyclonal anti-TSST-1 IgG (LTI-101), rabbitpolyclonal anti-TSST-1 IgG conjugated to horseradish peroxidase(LTC-101), and normal rabbit serum (NRS) certified anti-TSST-1 free(NRS-10) were purchased from Toxin Technology (Sarasota, Fla.). A 10microgram/millimeter solution of the polyclonal rabbit anti-TSST-1 IgGwas prepared in phosphate buffered saline (PBS) (pH 7.4). The PBS wasprepared from 0.016 molar NaH₂PO₄, 0.004 molar NaH₂PO₄—H₂O, 0.003 molarKCl and 0.137 molar NaCl, (Sigma Chemical Company, St. Louis, Mo.). Onehundred microliters of the polyclonal rabbit anti-TSST-1 IgG solutionwas pipetted into the inner wells of polystyrene microplates(Nunc-Denmark, Catalogue Number 439454). The plates were covered andincubated at room temperature overnight. Unbound anti-toxin was removedby draining until dry. TSST-1 was diluted to 10 nanograms/milliliter inPBS with phosphate buffered saline (pH7.4) containing 0.05% (vol/vol)Tween-20 (PBS-Tween) (Sigma Chemical Company, St. Louis, Mo.) and 1% NRS(vol/vol) and incubated at 4° C. overnight. Test samples were combinedwith 1% NRS (vol/vol) and incubated at 4° C. overnight. The plates weretreated with 100 microliters of a 1% solution of the sodium salt ofcasein in PBS (Sigma Chemical Company, St. Louis, Mo.), covered andincubated at 35° C. for one hour. Unbound BSA was removed by 3 washeswith PBS-Tween. TSST-1 reference standard (10 nanograms/milliliter)treated with NRS, test samples treated with NRS, and reagent controlswere pipetted in 200 microliter volumes to their respective wells on thefirst and seventh columns of the plate. One hundred microliters ofPBS-Tween was added to the remaining wells. The TSST-1 referencestandard and test samples were then serially diluted 6 times in thePBS-Tween by transferring 100 microliters from well-to-well. The sampleswere mixed prior to transfer by repeated aspiration and expression. Thiswas followed by incubation for 1.5 hours at 35° C. and five washes withPBS-T and three washes with distilled water to remove unbound toxin. Therabbit polyclonal anti-TSST-1 IgG conjugated to horseradish peroxidasewash diluted according to manufacturer's instructions and 50 microliterswas added to each microtiter well, except well A-1, the conjugatecontrol well. The plates were covered and incubated at 35° C. for onehour.

[0088] Following incubation the plates were washed five times inPBS-Tween and three times with distilled water. Following the washes,the wells were treated with 100 microliters of horseradish peroxidasesubstrate buffer consisting of 5 milligrams of o-phenylenediamine and 5microliters of 30% hydrogen peroxide in 11 mL of citrate buffer (pH5.5). The citrate buffer was prepared from 0.012 M anhydrous citric acidand 0.026 molar dibasic sodium phosphate. The plates were incubated for15 minutes at 35° C. The reaction was stopped by the addition of 50microliters of a 5% sulfuric acid solution. The intensity of the colorreaction in each well was evaluated using the BioTek Model EL309microplate reader (OD 490 nanometers). TSST-1 concentrations in the testsamples were determined from the reference toxin regression equationderived during each assay procedure. The efficacy of the compound ininhibiting the production of TSST-1 is shown in Table I below.

[0089] In accordance with the present invention, the data in Table 1shows that S. aureus (MN8), when compared to the control, producedsignificantly less TSST-1 in the presence of the aromatic compounds. Thearomatic compounds reduced the amount of exotoxin production rangingfrom about 91% to about 96%. However, although the amount of toxinproduced was significantly reduced, there was minimal, if any, effect onthe growth of S. aureus cells. TABLE 1 ELISA: TSST-1 Reduction % TestOptical ng/OD of Toxin Compound Compound Density CFU/mL unit (%) GrowthZero 0.625 2.8E+08 1504 N/A Medium Methanol 400 μL 0.627 2.8E+08 1440N/A Phenylethyl 0.5% 0.542 1.6E+08 60 96% alcohol Benzyl alcohol 0.5%0.792 1.8E+08 131 91% 2- 1.0% 0.549 9.0E+07 65 95% hydroxy- benzamide

EXAMPLE 2

[0090] In this Example, the effect of various test compounds on thegrowth of S. aureus and the production of TSST-1 was determined. Theeffect of the test compounds tested in Example 2 was determined byplacing the desired concentration, expressed in percent of the activecompound, in 10 mL of a growth medium as described in Example 1. Thetest compounds were then tested and evaluated as in Example 1.

[0091] In accordance with the present invention, Table 2 shows that S.aureus (MN8), when compared to the control, produced significantly lessTSST-1 in the presence of the aromatic compounds. The aromatic compoundsreduced the amount of exotoxin production ranging from about 82% to 97%.However, although the amount of toxin produced was significantlyreduced, there was minimal, if any, effect on the growth of S. aureuscells. TABLE 2 ELISA: Reduc- % Test TSST-1 tion of Com- Optical ng/ODToxin Compound pound Density CFU/mL unit % Growth Medium Zero0.607 >1.6E+09 2424 N/A Methanol 400 μL 0.598   2.6E+09 2690 N/APhenylethyl 0.5% 0.551   4.2E+08 68 97% alcohol Phenoxyethanol 0.6%0.681   8.3E+08 70 97% Phenoxyethanol 0.5% 0.728 >1.7E+09 122 95% p-0.2% 0.356 >1.5E+08 506 82% hydroxybenzoic acid, methyl ester 2- 0.2%0.682   1.48E+09 193 93% hydroxybenzoic acid p-aminobenzoic 0.2% 0.618  1.1E+09 317 89% acid

EXAMPLE 3

[0092] In this Example, the effect of various test compounds on thegrowth of S. aureus and the production of TSST-1 was determined. Theeffect of the test compounds tested in Example 3 was determined byplacing the desired concentration, expressed in percent of the activecompound, in 10 mL of a growth medium as described in Example 1. Thetest compounds were then tested and evaluated as in Example 1.

[0093] In accordance with the present invention, Table 3 shows that S.aureus (MN8), when compared to the control, produced significantly lessTSST-1 in the presence of the aromatic compounds. The aromatic compoundsreduced the amount of exotoxin production ranging from about 69% to 98%.However, although the amount of toxin produced was significantlyreduced, there was minimal, if any, effect on the growth of S. aureuscells. TABLE 3 ELISA: % Test TSST-1 Com- Optical ng/OD ReductionCompound pound Density CFU/mL unit of Toxin % Growth Medium Zero 0.6273.9E+09 1931 N/A Methanol 100 μL 0.588 5.2E+09 2041 N/A Phenylethyl 0.5%0.476 5.5E+08 46 98% alcohol Trans- 0.5% 0.549 1.7E+09 436 82% cinnamicacid Acetyl 0.5% 0.549 1.7E+09 436 69% tyrosine Gallic acid 0.5% 0.4921.2E+ 09 63 95%

EXAMPLE 4

[0094] In this Example, the effect of various test compounds on thegrowth of S. aureus and the production of TSST-1 was determined. Theeffect of the test compounds tested in Example 4 was determined byplacing the desired concentration, expressed in percent of the activecompound, in 10 mL of a growth medium as described in Example 1. Thetest compounds were then tested and evaluated as in Example 1.

[0095] In accordance with the present invention, Table 4 shows that S.aureus (MN8), when compared to the control, produced significantly lessTSST-1 in the presence of the aromatic compounds. The aromatic compoundsreduced the amount of exotoxin production ranging from about 79% to 98%.However, although the amount of toxin produced was significantlyreduced, there was minimal, if any, effect on the growth of S. aureuscells. TABLE 4 ELISA: % Test TSST-1 Com- Optical ng/OD ReductionCompound pound Density CFU/mL unit of Toxin % Growth Medium Zero 0.6063.2E+09 1445 N/A Methanol 100 μL 0.567 1.3E+09 1151 N/A Phenylethyl 0.5%0.554 5.4E+08 25 98% alcohol 4- 0.5% 0.629 2.4E+09 230 79%Acetamidophenol

EXAMPLE 5

[0096] In this Example the growth of S. aureus and the production ofTSST-1 in the presence of phenylethyl alcohol was measured usingdifferent TSST-1 producing strains of S. aureus. S. aureus FRI-1187 andFRI-1169 were obtained as lyophilized cultures from the stock collectionof Dr. Merlin Bergdoll, Food Research Institute (Madison Wis.). Theeffect of the phenylethyl alcohol was determined by placing the desiredconcentration, expressed in percent of the active compound, in 10 mL ofa growth medium as in Example 1. The phenylethyl alcohol was then testedand evaluated as in Example 1.

[0097] In accordance with the present invention, Table 5 shows that S.aureus when compared to the control, produced significantly less TSST-1in the presence of the phenylethyl alcohol. The phenylethyl alcoholreduced the amount of exotoxin production from the FRI-1169 culture fromabout 95% to about 100%. The phenylethyl alcohol also significantlyreduced the amount of exotoxin production from the FRI-1187 culture.However, although the amount of toxin produced was significantlyreduced, there was minimal, if any, effect on the growth of S. aureuscells. TABLE 5 ELISA: TSST-1 % Test Optical ng/OD Reduction CompoundCompound Density CFU/mL unit of Toxin % S. aureus FRI-11698 Growth Zero1.068 1.11e+09 158 N/A medium Phenylethyl  0.5% 1.263 3.03E+08 2  99%alcohol Phenylethyl 0.25% 1.208 2.05E+09 8  95% alcohol S. aureusFRI-1187 Growth Zero 1.056 1.59E+09 92 N/A medium Phenylethyl  0.5%1.296 2.55E+08 none 100% alcohol detected Phenylethyl 0.25% 1.2441.80E+09 1  98% alcohol

EXAMPLE 6

[0098] In this Example, the effect of test compounds in combination withsurface active agents was evaluated utilizing a checkerboardexperimental design. This allowed the evaluation of the interaction oftwo test compounds on the growth of S. aureus and the production ofTSST-1. Four concentrations of one test compound (including zero) werecombined with five concentrations of a second test compound (includingzero) in test tubes. In this Example, phenyethyl alcohol (0%, 0.5%,0.3%, 0.15%, and 0.05%) was combined with Cetiol 1414E (myreth-3myristate) (10 mM, 5 mM, 2.5 mM and 0). The test solutions wereotherwise prepared as described in Example 1 and evaluated in the samemanner as Example 1.

[0099] As Table 6 below indicates, at every concentration of Cetiol1414E, the phenylethyl alcohol increased the inhibition of production ofTSST-1, and vice versa. The effect appears to be additive. TABLE 6Cetiol ng TSST- ng TSST-1 Reduction 1414E PEA (%) 1/mL CFU/mL Log CFU/mLper CFU of Toxin %   0 0.5 106 3.95E+08 8.6 27 93%   0 0.3 201 5.15E+088.7 39 90%   0 0.15 561 4.35E+08 8.6 129 67%   0 0.05 826 3.10E+08 8.5266 32%   0 0 1178 3.00E+08 8.5 393  0%  10 mM 0.5 20 4.70E+08 8.7 4 99% 10 mM 0.3 59 7.20E+08 8.9 8 98%  10 mM 0.15 137 4.30E+08 8.6 32 92%  10mM 0.05 240 4.60E+08 8.7 52 87%  10 mM 0 262 4.30E+08 8.6 61 84%   5 mM0.5 58 6.25E+08 8.8 9 98%   5 mM 0.3 155 4.00E+08 8.6 39 90%   5 mM 0.15348 4.10E+08 8.6 85 78%   5 mM 0.05 538 4.75E+08 8.7 113 71%   5 mM 0558 3.25E+08 8.5 172 56% 2.5 mM 0.5 76 6.90E+08 8.8 11 97% 2.5 mM 0.3197 2.80E+08 8.4 70 82% 2.5 mM 0.15 384 4.95E+08 8.7 78 80% 2.5 mM 0.05618 4.15E+08 8.6 149 62% 2.5 mM 0 765 3.20E+08 8.5 239 39%

EXAMPLE 7

[0100] In this Example, the effect of phenylethyl alcohol and4-hydroxybenzoic acid, methyl ester on the production of alpha-toxinfrom S. aureus strain RN 6390 was evaluated utilizing a standardhemolytic assay.

[0101] The S. aureus alpha-toxin is a hemolytic exoprotein that causestarget cell membrane damage and cell death. It is produced underenvironmental conditions similar to those seen with TSST-1 production.The effect of the test compounds on the growth of S. aureus and theproduction of alpha-toxin was carried out by placing the desiredconcentrations, expressed in percent of the active compound, in 100 mLof growth medium in 500 mL fleakers capped with aluminum foil. Thegrowth medium and inoculum were prepared as described in Example 1. Thefleakers were incubated in a 37° C. water bath with a gyratory shakerset at 180 rpm. Growth was followed by periodic optical densitymeasurements at 600 nm. When the growth obtained an optical density of1.0, 10 mL aliquots were removed for analysis. Plate counts wereperformed on the aliquots to determine cell count and culture purity.The remaining culture fluid was centrifuged at 2500 rpm for 15 minutesand the resulting supernatant filter sterilized and frozen at −70° C.until assayed.

[0102] Defibrinated rabbit red blood cells (Hema Resources, Aurora,Oreg.) were washed 3 times in Tris-saline buffer and re-suspended to aconcentration of 0.5% (volume/volume). The Tris-saline buffer consistedof 50 mM Trizma® hydrochloride/Trizma base and 100 mM sodium chloride,with a final pH of 7.0. Culture supernatants were serially diluted inTris-saline buffer from 1:2 to 1:256. One hundred microliters of eachdilution was added to nine hundred microliters of the rabbit red bloodcells. Each dilution was set up in triplicate. The tubes were incubatedfor 30 minutes at 37° C. The samples were then centrifuged at 800×g for6 minutes. Two two-hundred microliter aliquots of each tube weretransferred to a microtiter plate and the optical density determined at410 nm. Control fluids used in place of the culture supernatantsincluded tris-saline buffer (zero lysis), 10% sodium dodecyl sulfate(100% lysis), and sterile growth medium containing the test compound.Units of activity are expressed as the reciprocal of the dilution ofeach test sample giving 50% lysis in samples that were adjusted to thesame initial optical density. As Tables 7 and 8 below indicate bothphenylethyl alcohol and 4-hydroxybenzoic acid methyl ester significantlyreduced production of the alpha toxin. TABLE 7 Hemolytic Test % TestEndpoint % Toxin Compound Compound 50% lysis Inhibition None 0 103 N/APhenylethyl 0.3%  3  97% alcohol Phenylethyl 0.4% None 100% alcoholDetected

[0103] TABLE 8 Hemolytic Test % Test Endpoint % Toxin Compound Compound50% lysis Inhibition None 0 265 N/A 4- 0.1% 79 70% hydroxybenzoic acidmethyl ester 4- 0.2% 16 94% hydroxybenzoic acid methyl ester

EXAMPLE 8

[0104] In this Example, the effect of phenylethyl alcohol in combinationwith Glucopon was evaluated utilizing a checkerboard experimentaldesign. This allowed the evaluation of the interaction of two testcompounds on the growth of S. aureus and the production of TSST-1.

[0105] Five concentrations of phenylethyl alcohol (0.5%, 0.3%, 0.15%,0.05%, and 0.0%) were combined with four concentrations of Glucopon (1.5mM, 0.75 mM, 0.25 mM and 0 mM) in a twenty tube array. For example, tube#1 contained 0 mM of Glucopon and 0.5% phenylethyl alcohol (vol/vol) in10 mL of growth medium (as prepared in Example 1). Each of tubes #1-#20contained a unique combination of Glucopon and phenylethyl alcohol.These combinations were tested and evaluated as in Example 1. The effectof the test compounds on the growth of S. aureus and on the productionof TSST-1 is shown in Table 9 below. TABLE 9 ng TSST- % Glucopon PEA (%)OD 1/OD CFU/mL Reduction   0 mM 0.0 0.685 755 9.05E+08 N/A   0 mM 0.050.712 323 1.07E+09 57%   0 mM 0.15 0.730 152 2.59E+09 80%   0 mM 0.30.758 54 1.97E+09 93%   0 mM 0.50 0.721 13 2.15E+09 98% 0.25 mM 0.00.660 542 1.26E+09 28% 0.25 mM 0.05 0.690 351 2.05E+09 54% 0.25 mM 0.150.705 173 2.44E+09 77% 0.25 mM 0.3 0.797 48 2.20e+09 94% 0.25 mM 0.50.657 14 1.21E+09 98% 0.75 mM 0.0 0.701 599 9.55E+08 21% 0.75 mM 0.050.705 285 8.60E+08 62% 0.75 mM 0.15 0.743 148 9.75E+08 80% 0.75 mM 0.30.731 45 2.19E+09 94% 0.75 mM 0.5 0.099 0 4.51E+07 100%  1.5 mM 0.00.718 196 1.83E+09 74%  1.5 mM 0.05 0.730 132 1.97E+09 83%  1.5 mM 0.150.694 68 1.11E+09 91%  1.5 mM 0.3 0.390 28 >5.00E+07   96%  1.5 mM 0.50.014 0 no N/A growth

[0106] As Table 9 below indicates, at every concentration of glucoponthe phenylethyl alcohol increased the inhibition of production ofTSST-1, and vice versa. The effect appears to be additive.

EXAMPLE 10

[0107] In this Example, the effect of Cetiol in combination withpara-aminobenzoic acid was evaluated utilizing a checkerboardexperimental design. This allowed the evaluation of the interaction oftwo test compounds on the growth of S. aureus and the production ofTSST-1.

[0108] Five concentrations of para-aminobenzoic acid (0.05%, 0.09%,0.19%, 0.38%, and 0.0%) were combined with four concentrations of Cetiol(2.5 mM, 5 mM, 10 mM and 0 mM) in a twenty tube array. For example, tube#1 contained 0% of para-aminobenzoic acid and 0 mM Cetiol (vol/vol) in10 mL of growth medium (as prepared in Example 1). Each of tubes #1-#20contained a unique combination of Cetiol and para-aminobenzoic acid.These combinations were tested and evaluated as in Example 1. The effectof the test compounds on the growth of S. aureus and on the productionof TSST-1 is shown in Table 10 below. TABLE 10 ng TSST- Cetiol PABA OD1/OD CFU/mL % Reduction   0 mM   0% 0.517 4907 8.90E+08 N/A   0 mM 0.05%0.546 5670 1.53E+09 0%   0 mM 0.09% 0.558 3389 1.85E+09 31%   0 mM 0.19%0.599 1975 1.79E+09 60%   0 mM 0.38% 0.589 1039 1.15E+09 79% 2.5 mM   0%0.637 3367 1.21E+09 31% 2.5 mM 0.05% 0.632 2193 1.89E+09 55% 2.5 mM0.09% 0.616 2413 1.46E+09 51% 2.5 mM 0.19% 0.611 2106 1.38E+09 57% 2.5mM 0.38% 0.612 891 1.31E+09 82%   5 mM   0% 0.881 2419 8.25E+08 51%   5mM 0.05% 0.957 1942 4.75E+08 60%   5 mM 0.09% 0.862 1875 8.25E+08 62%  5 mM 0.19% 0.849 1048 8.90E+08 79%   5 mM 0.38% 0.971 221 1.19E+09 95% 10 mM   0% 0.976 2286 3.95E+08 53%  10 mM 0.05% 1.317 1420 4.80E+08 71% 10 mM 0.09% 1.266 1244 8.10E+08 75%  10 mM 0.19% 0.806 674 6.00E+08 86% 10 mM 0.38% 0.749 467 6.55E+08 90%

[0109] In view of the above, it will be seen that the several objects ofthe invention are achieved. As various changes could be made in theabove-described non-absorbent articles without departing from the scopeof the invention, it is intended that all matter contained in the abovedescription be interpreted as illustrative and not in a limiting sense.

What is claimed is:
 1. An exoprotein inhibitor for inhibiting theproduction of exoproteins from Gram positive bacteria in and around thevagina comprising a non-absorbent substrate for insertion into a vaginabeing selected from the group consisting of a non-absorbent incontinencedevice, a barrier birth control device, a tampon applicator, and adouche, the non-absorbent substrate having deposited thereon aneffective amount of a first active ingredient having the generalformula:

wherein R¹ is —OR⁶OH; R⁶ is a divalent saturated or unsaturatedaliphatic hydrocarbyl moiety; R², R³, and R⁴ are independently selectedfrom the group consisting of H, OH, COOH, and —C(O)R⁹; R⁹ is hydrogen ora monovalent saturated or unsaturated aliphatic hydrocarbyl moiety,wherein the first active ingredient is effective in inhibiting theproduction of exoprotein from Gram positive bacteria.
 2. The exoproteininhibitor as set forth in claim 1 wherein R⁶ is a divalent saturated orunsaturated aliphatic hydrocarbyl moiety having from 1 to about 15carbon atoms.
 3. The exoprotein inhibitor as set forth in claim 2wherein R⁶ is a divalent saturated or unsaturated aliphatic hydrocarbylmoiety having from 1 to about 10 carbon atoms.
 4. The exoproteininhibitor as set forth in claim 2 wherein R⁶ is a divalent saturated orunsaturated aliphatic hydrocarbyl moiety having from 1 to about 6 carbonatoms.
 5. The exoprotein inhibitor as set forth in claim 1 wherein R² isOH and R³ is COOH.
 6. The exoprotein inhibitor as set forth in claim 1wherein the first active ingredient is phenoxyethanol.
 7. The exoproteininhibitor as set forth in claim 1 wherein the first active ingredient ispresent in an amount of at least about 0.01 micromoles per gram ofnon-absorbent substrate.
 8. The exoprotein inhibitor as set forth inclaim 1 wherein the first active ingredient is present in an amount fromabout 0.5 micromoles per gram of non-absorbent substrate to about 100micromoles per gram of non-absorbent substrate.
 9. The exoproteininhibitor as set forth in claim 1 wherein the first active ingredient ispresent in an amount from about 1.0 micromoles per gram of non-absorbentsubstrate to about 50 micromoles per gram of non-absorbent substrate.10. The exoprotein inhibitor as set forth in claim 1 further comprisinga pharmaceutically active material selected from the group consisting ofantimicrobials, antioxidants, anti-parasitic agents, antipruritics,astringents, local anaesthetics and anti-inflammatory agents.
 11. Theexoprotein inhibitor as set forth in claim 1 further comprising aneffective amount of a second active ingredient, said second activeingredient comprising a compound with an ether, ester, amide,glycosidic, or amine bond linking a C₈-C₁₈ fatty acid to an aliphaticalcohol wherein the second active ingredient is effective insubstantially inhibiting the production of exoprotein from Gram positivebacteria.
 12. The exoprotein inhibitor as set forth in claim 11 whereinthe C₈-C₁₈ fatty acid is linked to a polyalkoxylated sulfate salt. 13.The exoprotein inhibitor as set forth in claim 11 wherein the C₈-C₁₈fatty acid is linked to a sulfosuccinic salt.
 14. The exoproteininhibitor as set forth in claim 1 further comprising an effective amountof a second active ingredient having the general formula:

wherein R¹⁰ is a straight or branched alkyl or straight or branchedalkenyl having from 8 to about 18 carbon atoms and R¹¹ is selected fromthe group consisting of an alcohol, a polyalkoxylated sulfate salt and apolyalkoxylated sulfosuccinate salt wherein the second active ingredientis effective in substantially inhibiting the production of exoproteinfrom Gram positive bacteria.
 15. The exoprotein inhibitor as set forthin claim 14 wherein R¹⁰ is a straight or branched alkyl group.
 16. Theexoprotein inhibitor as set forth in claim 14 wherein R¹⁰ is a straightor branched alkenyl group.
 17. The exoprotein inhibitor as set forth inclaim 14 wherein R¹⁰ is obtained from the group consisting of caprylicacid, capric acid, lauric acid, myristic acid, palmitic acid and stearicacid.
 18. The exoprotein inhibitor as set forth in claim 14 wherein R¹¹is an aliphatic alcohol.
 19. The exoprotein inhibitor as set forth inclaim 18 wherein R¹¹ is an aliphatic alcohol selected from the groupconsisting of glycerol, glycol, sucrose, glucose, sorbitol, andsorbitan.
 20. The exoprotein inhibitor as set forth in claim 19 whereinR¹¹ is a glycol selected from the group consisting of ethylene glycol,propylene glycol, polypropylene glycol, and combinations thereof. 21.The exoprotein inhibitor as set forth in claim 14 wherein the secondactive ingredient is selected from the group consisting of laureth-3,laureth-4, laureth-5, PPG-5 lauryl ether, 1-0-dodecyl-rac-glycerol,sodium laureth sulfate, potassium laureth sulfate, disodium laureth (3)sulfosuccinate, dipotassium laureth (3) sulfosuccinate and polyethyleneoxide (2) sorbitol ether.
 22. The exoprotein inhibitor as set forth inclaim 14 wherein the second active ingredient is present in an amount ofat least about 0.0001 millimoles per gram of non-absorbent substrate.23. The exoprotein inhibitor as set forth in claim 14 wherein the secondactive ingredient is present in an amount of at least about 0.005millimoles per gram of non-absorbent substrate.
 24. The exoproteininhibitor as set forth in claim 14 wherein the second active ingredientis present in an amount from about 0.005 millimoles per gram ofnon-absorbent substrate to about 0.2 millimoles per gram ofnon-absorbent substrate.
 25. The exoprotein inhibitor as set forth inclaim 14 further comprising a pharmaceutically active material selectedfrom the group consisting of antimicrobials, antioxidants,anti-parasitic agents, antipruritics, astringents, local anaestheticsand anti-inflammatory agents.
 26. The exoprotein inhibitor as set forthin claim 1 further comprising an effective amount of a second activeingredient, the second active ingredient comprising an alkylpolyglycoside effective in substantially inhibiting the production ofexoprotein from Gram positive bacteria.
 27. The exoprotein inhibitor asset forth in claim 26 wherein the alkyl polyglycoside has an alkyl grouphaving from about 8 to about 18 carbon atoms.
 28. The exoproteininhibitor as set forth in claim 27 wherein the alkyl group is a linearalkyl group.
 29. The exoprotein inhibitor as set forth in claim 27wherein the alkyl polyglycoside has an alkyl group having from about 8to about 14 carbon atoms.
 30. The exoprotein inhibitor as set forth inclaim 26 wherein the alkyl polyglycoside has an HLB of 12 to
 14. 31. Theexoprotein inhibitor as set forth in claim 26 wherein the alkylpolyglycoside has an HLB of 10 to
 15. 32. The exoprotein inhibitor asset forth in claim 26 wherein the alkyl polyglycoside has the generalformula:

wherein Z is a saccharide residue having 5 or 6 carbon atoms, n is awhole number from 1 to 6, and R¹⁴ is a linear alkyl group having fromabout 8 to about 18 carbon atoms.
 33. The exoprotein inhibitor as setforth in claim 32 wherein R¹⁴ is a linear alkyl group having from about8 to about 14 carbon atoms.
 34. The exoprotein inhibitor as set forth inclaim 32 wherein R¹⁴ is a linear alkyl group having from about 8 toabout 12 carbon atoms.
 35. The exoprotein inhibitor as set forth inclaim 26 wherein the second active ingredient is present in an amount ofat least about 0.0001 millimoles per gram of non-absorbent substrate.36. The exoprotein inhibitor as set forth in claim 26 wherein the secondactive ingredient is present in an amount of at least about 0.005millimoles per gram of non-absorbent substrate.
 37. The exoproteininhibitor as set forth in claim 26 wherein the second active ingredientis present in an amount of at least about 0.005 millimoles per gram ofnon-absorbent substrate to about 2 millimoles per gram of non-absorbentsubstrate.
 38. The exoprotein inhibitor as set forth in claim 26 whereinthe alkyl polyglycoside is selected from the group consisting ofGlucopon 220, Glucopon 225, Glucopon 425, Glucopon 600, Glucopon 625,and TL
 2141. 39. The exoprotein inhibitor as set forth in claim 1further comprising an effective amount of a second active ingredientselected from the group consisting of glycerol monolaurate andmyreth-3-myristate wherein said active ingredient is effective insubstantially inhibiting the production of exoprotein from Gram positivebacteria.
 40. The exoprotein inhibitor as set forth in claim 1 furthercomprising an effective amount of a second active ingredient having thegeneral formula:

wherein R¹⁷, inclusive of the carbonyl carbon, is an alkyl group having8 to 18 carbon atoms, and R¹⁸ and R¹⁹ are independently selected fromhydrogen or an alkyl group having from 1 to about 12 carbon atoms whichmay or may not be substituted with groups selected from ester groups,ether groups, amine groups, hydroxyl groups, carboxyl groups, carboxylsalts, sulfonate groups, sulfonate salts, and mixtures thereof whereinsaid second active ingredient is effective in substantially inhibitingthe production of exoprotein from Gram positive bacteria.
 41. Theexoprotein inhibitor as set forth in claim 40 wherein R¹⁷ is derivedfrom a saturated or unsaturated fatty acid.
 42. The exoprotein inhibitoras set forth in claim 41 wherein R¹⁷ is derived from an acid selectedfrom the group consisting of caprylic acid, capric acid, lauric acid,myristic acid, palmitic acid, and stearic acid.
 43. The exoproteininhibitor as set forth in claim 40 wherein the second active ingredientis selected from the group consisting of sodium lauryl sarcosinate,lauramide monoethanolamide, lauramide diethanolamide, lauramidopropyldimethylamine, disodium lauramide monoethanolamide sulfosuccinate, anddisodium lauroamphodiacetate.
 44. The exoprotein inhibitor as set forthin claim 40 wherein the second active ingredient is present in an amountof at least about 0.0001 millimoles per gram of non-absorbent substrate.45. The exoprotein inhibitor as set forth in claim 40 wherein the secondactive ingredient is present in an amount of at least about 0.0005millimoles per gram of non-absorbent substrate.
 46. The exoproteininhibitor as set forth in claim 40 wherein the second active ingredientis present in an amount from about 0.005 millimoles per gram ofnon-absorbent substrate to about 0.2 millimoles per gram ofnon-absorbent substrate.
 47. The exoprotein inhibitor as set forth inclaim 40 further comprising a pharmaceutically active material selectedfrom the group consisting of antimicrobials, antioxidants,anti-parasitic agents, antipruritics, astringents, local anaestheticsand anti-inflammatory agents.
 48. The exoprotein inhibitor as set forthin claim 1 further comprising an effective amount of a second activeingredient having the general formula:

wherein R²⁰ is an alkyl group having from about 8 to about 18 carbonatoms and R²¹ and R²² are independently selected from the groupconsisting of hydrogen and alkyl groups having from 1 to about 18 carbonatoms and which can have one or more substitutional moieties selectedfrom the group consisting of hydroxyl, carboxyl, carboxyl salts andimidazoline wherein the second active ingredient is effective insubstantially inhibiting the production of exoprotein from Gram positivebacteria.
 49. The exoprotein inhibitor article as set forth in claim 48wherein R²² comprises a carboxyl salt, the carboxyl salt having acationic moiety selected from the group consisting of sodium, potassiumand combinations thereof.
 50. The exoprotein inhibitor as set forth inclaim 48 wherein R²² comprises an amine selected from the groupconsisting of lauramine, lauramino, propionic acid, sodiumlauriminodipropionic acid, lauryl hydroxyethyl imidazoline and mixturesthereof.
 51. The exoprotein inhibitor as set forth in claim 48 whereinthe second active ingredient is present in an amount of at least about0.0001 millimoles per gram of non-absorbent substrate.
 52. Theexoprotein inhibitor as set forth in claim 48 wherein the second activeingredient is present in an amount of at least about 0.005 millimolesper gram of non-absorbent substrate.
 53. The exoprotein inhibitor as setforth in claim 48 wherein the second active ingredient is present in anamount from about 0.005 millimoles per gram of non-absorbent substrateto about 0.2 millimoles per gram of non-absorbent substrate.
 54. Theexoprotein inhibitor as set forth in claim 48 further comprising apharmaceutically active material selected from the group consisting ofantimicrobials, antioxidants, anti-parasitic agents, antipruritics,astringents, local anaesthetics and anti-inflammatory agents.
 55. Theexoprotein inhibitor as set forth in claim 1 further comprising aneffective amount of a second active ingredient having the generalformula:

wherein R²³ is an alkyl group having from 8 to about 18 carbon atoms andR²⁴, R²⁵, and R²⁶ are independently selected from the group consistingof hydrogen and alkyl group having from 1 to about 18 carbon atoms andwhich can have one or more substitutional moieties selected from thegroup consisting of hydroxyl, carboxyl, carboxyl salts, and imidazolinewherein the second active ingredient is effective in substantiallyinhibiting the production of exoprotein from Gram positive bacteria. 56.The exoprotein inhibitor as set forth in claim 55 wherein the secondactive ingredient is triethanolamide laureth sulfate.
 57. The exoproteininhibitor as set forth in claim 55 wherein the second active ingredientis present in an amount of at least about 0.0001 millimoles per gram ofnon-absorbent substrate.
 58. The exoprotein inhibitor as set forth inclaim 55 wherein the second active ingredient is present in an amount ofat least about 0.005 millimoles per gram of non-absorbent substrate. 59.The exoprotein inhibitor as set forth in claim 55 wherein the secondactive ingredient is present in an amount from about 0.005 millimolesper gram of non-absorbent substrate to about 0.2 millimoles per gram ofnon-absorbent substrate.
 60. The exoprotein inhibitor as set forth inclaim 55 further comprising a pharmaceutically active material selectedfrom the group consisting of antimicrobials, antioxidants,anti-parasitic agents, antipruritics, astringents, local anaestheticsand anti-inflammatory agents.